The chain hemoglobinopathies such as sickle cell disease and thalassemia are among the most common inherited diseases in humans. Reactivation of chain expression can cure the diseases by compensating for the loss of the chain activity. The identification of transcription activators that activate globin gene expression and understanding the mechanisms by which they activate globin gene expression will provide new targets and strategies for the treatment of sickle cell disease and thalassemia. The long-term objectives of this proposal are to validate the in vivo role of the candidate globin transactivators FKLF1 and FKLF2 in globin gene expression, study the mechanisms by which they activate globin gene expression and identify transcription factors that activate endogenous globin expression through the CACCC box of the promoter. Our specific aims are: 1) To determine the in vivo role of FKLF1 and FKLF2 in globin gene activation; 2) To characterize FKLF1 and FKLF2 proteins; 3) To study the regulation of FKLF1 and FKLF2 transcriptional activation of globin gene by acetylation; 4) To study the regulation of FKLF1 and FKLF2 transcriptional activation of globin gene by phosphorylation; 5) To identify target genes of FKLF1 and FKLF2 in fetal erythroid cells; 6) To study the regulation of FKLF1 and FKLF2 gene expression; 7) If the studies in specific aim 1 show that FKLF1 or FKLF2 can not activate endogenous globin in vivo, we will then clone new factors that interact with the CACCC box and activate the globin gene. We will use the experimental approaches based on cell culture, genetic assays and molecular and biochemical methods to achieve our goals.